Protein switches Many proteins involved in cellular signal transduction switch between inactive and active conformations upon binding or release of ligands. In Escherichia coli, AraC protein is involved in regulation of the expression of genes whose products enable the cells to take up and catabolize the sugar, L-arabinose. Upon binding of arabinose AraC undergoes a conformational change and actively represses its own synthesis and the synthesis of the AraB, AraA, and AraD gene products. Certain mutations can render the protein unresponsive to binding of arabinose. It is not clear from experiments why this should be the case, especially if a hydrophobic residue is replaced with another hydrophobic residue. Our preliminary simulations of this protein with the Self-guided Langevin dynamics method in CHARMM have provided explanation of this behavior. We will simulate numerous mutants of this protein to understand further its behavior, and find ways to manipulate it. Any variants that appear interesting in simulations will be synthesized and examined in detail experimentally in the lab of our collaborator, Prof. Robert Schleif. The majority of the simulations will be run on the Open Science Grid through the recently implemented version of CHARMM on the Open Science Grid. Coupling between ionization of internal groups and protein conformational rearrangement Ionization of internal groups in proteins is at the core of energy transduction in biological systems. The ionization can trigger conformational rearrangements, which in turn can change the pKa values of ionizable groups. To study how the protein responds to the ionization of internal groups, we have performed molecular dynamics simulations of eighteen variants of staphylococcal nuclease (SN) in which ionizable groups are buried in the protein core. The work was performed in collaboration with Prof. Bertrand Garcia-Moreno at Johns Hopkins University, who has experimentally characterized a large number of variants of SN. Our results show how the ionization of internal groups can lead to significant increases in hydration and changes in the conformation of the internal side chains and to localized backbone relaxation. These findings will guide the development of accurate methods for structure-based pKa calculations. Calcium ATPase Conformational Transition through Self-Guided Langevin Dynamics Simulation The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) transport calcium ions from cytoplasm into the reticulum and relaxes the muscle cells. Many crystal structures of SERCA1 in various binding states have been determined, which provide insights into the mechanism of transport Ca2+ across the membrane. Molecular modeling and simulation studies are also devoted to the understanding of this important process. SERCA1a is an integral membrane protein. It comprises a single polypeptide chain of 994 amino acid residues. It is clear from the crystal structures that SERCA has a 10 helices trans-membrane domain (M), an actuator domain (A), a nucleotide binding domain (N), and a phosphorylation domain (P). The Ca2+ transport cycle starts with Ca2E1 through the Ca2+ dependent phosphorylation by ATP, leading to the formation of the Ca2E1P high-energy intermediate. Ca2E1P transits to Ca2E2P, which releases Ca2+ into the lumen of SR and leads to the E2P state. After dephosphorylation, E2P transits to E2 state and closes the luminal gate. Through thermo agitation, E2 transits to E1 by releasing protons into the cytoplasm. E1 has high Ca2+ affinity and binds with Ca2+ to form Ca2E1. To understand the transport mechanism, it is desirable to study the dynamic process during the conformation transition. Self-guided Langevin dynamics (SGLD) is a simulation method capable of studying events with large conformational change. SGLD simulations of SERCA at different binding states produce conformational transitions between conformational states. New conformations for E1.2Ca2+ and E2.P state have been identified and at E2 state the crystal structure is a preferred conformation. Application of replica path calculations in enzymatic reaction mechanism of matrix metalloproteinase-2 (MMP2) We are continuing our effort in applying advanced replica path methods developed in our lab to explore reaction mechanism in more enzymatic systems, including the inhibition mechanism of matrix metalloproteinase-2 (MMP2) by its highly selective inhibitor (SB-3CT). MMP2 serves as selective inhibitor design target during the past decade due to its unregulated activities in many human diseases. The MMP2 inhibition mechanism of SB-3CT remains unclear until recent experimental and theoretical discoveries. Various replica path methods available in CHARMM are applied to map out the the reaction paths reflecting the MMP2 inhibition mechanism of SB-3CT which involves coupling between three membered thiirane ring opening and proton transferring. These calculations demonstrated the power of our reaction path methods in enzymatic reaction mechanism studies. Structure and Reaction Mechanisms of Boronic Acids Two specific areas of boronic acid (BA) research are ongoing;the design of BA based synthetic receptors and the discovery of chemical mechanisms for BA based proteasome inhibitors. This effort has elucidated the experimentally suggested reaction mechanism for the metabolism of the proteasome inhibitor, bortezomib. Despite this success, metabolism of bortezomib requires further clarification as it may play a significant role in the growing chemoresistance of cancer cells to this novel boronic acid inhibitor. In collaboration with Tony James (Bath University, UK), John S. Fossey (University of Birmingham, UK) and Charles W. Bock (Philadelphia University), development of BA based sensors have led to a publication characterizing the fundamental chemical interaction that modulates fluorescence when these synthetic receptors are bound to saccharides. Results are to be further extended to the future design of sensors for glycoprotein detection and identification. Cell surface saccharides function as identity tags and mark specific diseases and the ability to detect these glyco-structures is an active area of research. BA based receptors possess the ability to be optimized for detection of a number of different glycosylated saccharides. Both of the above areas of research will be studied via QM and QM/MM methods making use of the MSCAle interface within CHARMM. Chemical mechanism studies will also use CHARMMs RPATh algorithm with the recently implemented availiabilty of holonomic constraints.